Structural And Functional Research On The Mechanism Of M₂⁶A-Dimethylated Modification For Conserved Stem-loop?h45?by TFB1M

Mitochondria are essential molecular machinery for the maintenance of cellular energy supply by the oxidative phosphorylation system(OXPHOS).Based on internal symbiosis origin theory,mitochondria contains a considerable amount of copies of circular double-stranded genome(mtDNA),which encodes the 13 essential peptides in endometrial complex of OXPHOS as well as ribosome RNAs,transfer RNAs in the transcriptional and translational machinery.Protein biogenesis in mitochondria is quite significant for the production of OXPHOS conponent.Differentiated to the elements in nucleus,the transcriptional machinery in mitocho ndria is composed of a bacterialphage-related RNA polymerase(POLRMT),transcription factor A with HMG-box and two transcription factors TFB1M and TFB2M.TFB1M and TFB2M are bifunctional homologous proteins,functioning as transcription factor and methyltransferase.However,TEBIM is identified as a methyltransferase,but TFB2M is involved in the formation of transcriptional initial complex as a transcriptio n factor.In the progre ss of bio genesis of mitochondrial transcriptional and translational machinery,the post-transcriptional modification in ribosome RNA participates in transcription,splicing,translation and ribosome functioa TFBIM is a dimethyltransferase and maintains mitochondrial homeostasis by catalyzing dimethylation of two adjacent adenines located in helix45(h45)of 12S rRNA.This unique m62A modification is currently only discovered in this location.TFB1M knock-out could lead to the embryo lethal in mouse.And deficiency of this protein cause the mitochondria dysfunction,including the low expression of 13 peptides in OXPHOS and decrease production of ATP,finally resulting in the desease.The deficiency of TFB1M is likely to be the pathogenesis of type 2 diabetes,due to the damage of demethylation and abnormal assembly of ribosome subunits.Therefore,m62A modification is indispensable for assembly and maturation of human mitochondrial ribosome s.In this work,using the method of X-ray crystallography,we recombined and purified human TFB1M protein in vitro,and also transcripted RNA helix45 using T7 phage RNA polymerase,reporting the crystal structures of a binary complex hsTFB1M-h45 and a ternary complex of human TFB1M--h45--S-adenosyl-methionine with resolution of 3.0A.The structures firstly revealed a distinct mode of hsTFB1M interaction with its rRNA substrate as well as the initial enzymatic state involved in m62A modification,showing m.937A was preferentially recognized by TFB1M and the pocket of this adenine was close to SAM.Mutants experiments of key residues on binding surface between TFB1M and helix45 revealed that binding between them depended on electrostatic interactions.Moreover,we analysed the enzyme activity by primer exte:nsion and proved mutations of enzyme active site or the binding interfice impeded the m62A modification of helix45.In hepatocellular carcinoma cells,suppression of hsTFBIM protein levelor overexpression of inactive hsTFBIM mutants resulted in decreased ATP production and reduced expression of components of the mitochondrial OXPHOS without affecting transcription of the corresponding genes and their localization to the mitochondria.SUnSET and OP-Puro expriments gave the further proof that deficiency of TFB1M inhibited the rate of mitochondrial protein synthesis.And we observed that mitochondrial small 28S subunit assembly reduced in deficiency TFBIM cells,but large 39S subunit was not affected.Thus,hsTFBIM regulated translation of mitocho ndrial genes rather than their transcription according to modify m62A in h45.In order to detect the conformational change of helix45 with and without m62A modification,we solved the solution structures by NMR method.The stem loop RNA presented a standard A-form fold in solution state and had a "GNRA" tetraloop.Compared the structure of RNA without m26A,methyl groups provided the space steric,but not affected the turning out of RNA.The tetraloop was more compact.Therefore,the conformational change of RNA mainly relied on TFB1M.